The present invention relates to new taxoids possessing strong antitumor activities, the precursors of these antitumor taxoids, and pharmaceutical compositions thereof.
Taxol (paclitaxel), a complex diterpene, is currently considered the most exciting lead in cancer chemotherapy. Paclitaxel possesses high cytotoxicity and strong antitumor activity against different cancers which have not been effectively treated by existing antitumor drugs. For example, paclitaxel has been approved by FDA in late 1992 for the treatment of advanced ovarian cancer and for breast cancer in 1994. Paclitaxel is currently in phase II and III clinical trial for lung cancer and other cancers.
Although paclitaxel is an extremely important xe2x80x9cleadxe2x80x9d in cancer chemotherapy, it is common that better drugs can be derived from naturally occurring lead compounds. In fact, French researchers have discovered that a modification of the C-13 side chain of paclitaxel brought about a new anticancer agent which seems to have antitumor activity superior to paclitaxel with better bioavailability. This synthetic compound was named xe2x80x9cTaxotxc3xa8re (docetaxel)xe2x80x9d, which has t-butoxycarbonyl instead of benzoyl on the amino group of (2R,3S)-phenylisoserine moiety at the C-13 position and a hydroxyl group instead of acetoxy group at C-10. Docetaxel is currently in phase II and III clinical trials in United States, Europe, and Japan, has shown excellent activity, especially against breast and lung cancers. 
A recent report on clinical trials of paclitaxel and docetaxel has disclosed that paclitaxel causes, e.g., nerve damage, muscle pain or disturbances in heart rhythm, whereas docetaxel provokes, e.g., mouth sores and a plunge in white blood cells. Other less serious side effects also exist for these two drugs. Therefore, it is very important to develop new anti-cancer drugs different from these two drugs which have fewer undesirable side effects, better pharmacological properties, improved activity against drug-resistant tumors, and/or activity spectra against various tumor types.
It is an objective of the present invention to develop such new anti-tumor agents of paclitaxel class, i.e., taxoids, which have distinct structural differences from those of paclitaxel and docetaxel.
It is an object of the present invention to provide a series of new taxoids bearing a 1-propenyl, 2-methyl-1-propenyl, 2-methylpropyl, or trifluromethyl radical at the C-3xe2x80x2 position instead of a phenyl group, and which possess strong antitumor activities with better therapeutic profile, in particular against drug-resistant tumors. One of the serious drawbacks of both paclitaxel and docetaxel is the fact that these two drugs possess only a weak activity against drug-resistant tumors, e.g., adriamycin-resistant breast cancer. The new taxoids of the present invention have shown not only stronger antitumor activities against human ovarian, non-small cell lung, colon, and breast cancers than those of the two drugs, but also exhibit more than one order of magnitude better activity against adriamycin-resistant human breast cancer cells than those of the two drugs. Multi-drug-resistance (MDR) is a serious issue in clinical oncology, and thus the new taxoid antitumor agents of this invention will serve as important drugs to overcome this problem.
One aspect of the invention is a taxoid of the formula (I) 
in which
R1 is a C3-C5 alkyl or alkenyl or trifluoromethyl radical;
R2 is a C3-C5 branched alkyl radical;
R3 and R4 are independently selected from hydrogen and hydroxyl protecting groups including functional groups which increase the water solubility of the taxoid antitumor agent;
R5 represents a hydrogen or hydroxyl-protecting an acyl or alkoxycarbonyl or carbamoyl group;
R6 represents an acyl radical,
which are useful as antitumor agents or their precursors.
Preferably, R1 is selected from propyl, 2-methyl-1-propenyl, 1-methyl-1-propenyl, 2-methylpropyl, 1-methylpropyl, tert-butyl, cyclopropyl, cyclopropylmethyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methylbutyl, 2-methylbutyl, isobutyl, 2-methylethyl, 3-methylbutyl, 2-butenyl, or trifluoromethyl radicals;
R2 is selected from isopropyl, cyclopropyl, isobutyl, sec-butyl, 2-methylpropyl, 3-methylpropyl, tert-butyl, cyclobutyl, cyclopentyl, 1-ethylpropyl, or 1,1-dimethylpropyl radicals;
R5 is selected from hydrogen, C2-C6 acyl, C1-C6 alkoxylcarbonyl, C1-C6 N-alkylcarbamoyl, or C1-C6 N,N-dialkylcarbamoyl radicals; and
R6 is selected from benzoyl, fluorobenzoyl, chlorobenzoyl, azidobenzoyl, cyclohexanecarbonyl, acryloyl, crotonoyl, 1-methylacryloyl, 2-methyl-2-butenoyl, or 3-methyl-3-butenoyl radical.
More preferably, R5 is selected from acetyl, propanoyl, cyclopropanecarbonyl, acryloyl, crotonoyl, 3,3-dimethylacryloyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, pyrrolidine-N-carbonyl, piperidine-N-carbonyl, morpholine-N-carbonyl, methoxycarbonyl, ethoxylcarbonyl, propoxylcarbonyl, butoxycarbonyl, cyclopentanecarbonyl, or cyclohexanecarbonyl radicals.
These new taxoids (I) are synthesized by the processes which comprise the coupling reactions of the baccatin of the formula (II) 
wherein G1 represents a hydroxyl protecting group, and R5 and R6 have been defined above, with the xcex2-lactams of the formula (III) 
wherein G is a hydroxyl protecting group such as ethoxyethyl (EE), triethylsilyl (TES), (tert-butyl)dimethylsilyl (TBS), and triisopropylsilyl (TIPS), and R1 and R2 have been defined above, in the presence of a base.
Another aspect of the invention is a taxoid of the formula (7): 
wherein
R1 is a branched or unbranched C3-C5 alkyl or alkenyl radical, CF2H, or (S)-2,2-dimethylcyclopropyl; R8 is a C1-C4 alkyl radical; and R7 is F, Cl, MeO, vinyl, Me, or N3.
R1 is preferably a branched or unbranched C4 alkyl or alkenyl radical, more preferably CH2CH(CH3)2 or CHxe2x95x90C(CH3)2. R7 is preferably MeO or N3.
In one embodiment, R1 is CH2CH(CH3)2, R7 is MeO, and R8 is ethyl. In another embodiment, R1 is CHxe2x95x90C(CH3)2, R7 is MeO, and R8 is ethyl. In yet another embodiment, R1 is CHxe2x95x90C(CH3)2, R7 is N3, and R8 is ethyl.
The invention also encompasses a method for treating tumors which comprises administering to a patient an effective amount of the taxoid of formula (7). Preferably, the method treats leukemia, melanoma, breast, non-small cell lung, ovarian, and colon cancers.
Yet another aspect of the invention is a pharmaceutical composition having antineoplastic activity containing the taxoid of formula (7) and a physiologically acceptable carrier.
A further aspect of the invention is a method for preparing a taxoid of formula (7), including coupling a baccatin of formula (5) with a xcex2-lactam of formula (6) in the presence of a base, 
wherein G and G1, which may be the same or different, each represents a hydroxyl protecting group, and R1, R7, and R8 are as defined for formula (7). Preferably, G and G1 are independently ethoxyethyl (EE), triethylsilyl (TES), (tert-butyl)dimethylsilyl (TBS), or triisopropylsilyl (TIPS).
New taxoids of the formula (I) hereinabove are useful as antitumor agents or their precursors. These taxoids possess strong antitumor activities against human breast, non-small cell lung, ovarian, and colon cancers including drug-resistant cancer cells, as well as leukemia and melanoma.
The new taxoids of the formula (I) are synthesized by modifying the baccatins of the formula (II) 
wherein G1, R5, and R6 have been defined above.
The baccatins (II) are coupled with the xcex2-lactamis of the formula (III) 
wherein G, R1, and R2 have been defined hereinabove, to yield the new taxoids (I).
The xcex2-lactams (III) are readily prepared via the xcex2-lactams (IV) which are easily obtained through the chiral enolate-imine cyclocondensation method that has been developed in the present inventor""s laboratory as shown in Scheme 1 (Ojima et al., Bioorg. Med. Chem. Lett., 1993, 3, 2479, Ojima et al., Tetrahedron Lett., 1993, 34, 4149, Ojima et al., Tetrahedron Lett. 1992, 33, 5739, Ojima et al., Tetrahedron, 1992, 48, 6985, Ojima, I. et al., J. Org. Chem., 1991, 56, 1681, the disclosures of which are incorporated herein by reference). In this preparation, the xcex2-lactams (IV) with extremely high enantiomeric purities are obtained in high yields. In Scheme 1, R* is a chiral auxiliary moiety which is (xe2x88x92)-trans-2-phenyl-1-cyclohexyl or (xe2x88x92)-10-dicyclohexylsulfamoyl-D-isobornyl, TMS is a trimethylsilyl radical, and the base is lithium diisopropylamide or lithium hexamethyldisilazide and G and R1 have been defined hereinabove. 
The xcex2-lactams (IV) can be converted to the corresponding N-alkoxycarbonyl-xcex2-lactams (III) in excellent yields by reacting with alkyl chloroformates in the presence of a base (Scheme 2). This transformation is known to those skilled in the art.
The xcex2-lactams (III) are readily used for coupling with the baccatins (II) in the presence of a base, followed by deprotection to give the new taxoids (I) in high yields (Scheme 3). 
In Schemes 1-3, R1 through R6 have been defined above, M is an alkali metal, and the hydroxyl protecting group G1 is independently selected from methoxylmethyl (MOM), methoxyethyl (MEM), 1-ethoxyethyl (EE), benzyloxymethyl, (xcex2-trimethylsilylethoxyl)-methyl, tetrahydropyranyl, 2,2,2-trichloroethoxylcarbonyl (Troc), benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl (Fmoc), 2,2,2-trichloroethoxymethyl, trimethylsilyl, triethylsilyl, tripropylsilyl, dimethylethylsilyl, dimethyl(t-butyl)silyl, diethylmethylsilyl, dimethylphenylsilyl, diphenylmethylsilyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl.
The coupling reaction of the baccatin (II) and the xcex2-lactams (VI) is carried out via an alkali metal alkoxide of the baccatin (II) at the C-13 hydroxyl group. The alkoxide can readily be generated by reacting the baccatin with an alkali metal base such as sodium hexamethyldisilazide, potassium hexamethyldisilazide, lithium hexamethyldisilazide, sodium diisopropylamide, potassium diisopropylamide, lithium diisopropylamide, sodium hydride, in a dry nonprotic organic solvent such as tetrahydrofuran (THF), dioxane, ether, dimethoxyethane (DME), diglyme, dimethylformamide (DMF), mixtures of these solvents with hexane, toluene, and xylene, in a preferred temperature range from about xe2x88x92100xc2x0 C. to about 50xc2x0 C., more preferably at about xe2x88x9278xc2x0 C. to about 25xc2x0 C. This reaction is preferably carried out under inert atmosphere such as nitrogen and argon. The amount of the base used for the reaction is preferably approximately equivalent to the amount of the baccatin when soluble bases such as sodium hexamethyldisilazide, potassium hexamethyldisilazide, lithium hexamethyldisilazide, sodium diisopropylamide, potassium diisopropylamide, lithium diisopropylamide are used. The use of a slight excess of the base does not adversely affect the reaction. When heterogeneous bases such as sodium hydride and potassium hydride are used, 5-10 equivalents of the base (to the amount of the baccatin) are preferably employed.
The coupling reaction of the metal alkoxide of the baccatin thus generated with the xcex2-lactam is typically carried out by adding the solution of the xcex2-lactam in a dry organic solvent exemplified above in a preferred temperature range from about xe2x88x92100xc2x0 C. to 50xc2x0 C., more preferably at about xe2x88x9235xc2x0 C. to 25xc2x0 C. The mixture of reactants is stirred for 15 minutes to 24 hours and the progress and the completion of the reaction is monitored by thin layer chromatography (TLC), for example. When the limiting reactant is completely consumed, the reaction is quenched by addition of a cold brine solution. The crude reaction mixture is worked up using the standard isolation procedures which are generally known to those skilled in the art to give the corresponding taxoid. The proportion of the xcex2-lactam and the baccatin is in a range from 2:1 to 1:2, more preferably approximately 1:1 for purposes of economy and efficiency, but the ratio is not critical for the reaction.
The hydroxyl protecting groups can then be removed by using the standard procedures which are generally known to those skilled in the art to give the desired taxoid derivatives. For example, EE and TES groups can be removed with 0.5 N HCl at room temperature for 36 h, TIPS and TBS groups can be removed by treating with fluoride ion or HF in a non-protic organic solvent, and Troc group can be removed with zinc and acetic acid in methanol at 60xc2x0 C. for 1 hour without disturbing the other functional groups and the skeleton of the taxoid.
It has been shown that the introduction of 2-methyl-1-propenyl group to the C-3xe2x80x2 position of paclitaxel appears to increase the cytotoxicity, especially against drug-resistant cancer cells: Holton and Nadizadeh disclosed in U.S. Pat. No. 5,284,864 (1994) that 3xe2x80x2-dephenyl-3xe2x80x2-isobutenylpaclitaxel (RAH-1) exhibited 4 times better activity than paclitaxel and 7 times better activity than docetaxel against human colon carcinoma cells HCT-116, and also about 20 times better activity than paclitaxel and 9 times better activity than docetaxel against multi-drug resistant phenotype human colon carcinoma cells HCT-116/VM.
We have found that the structural requirements for taxoid antitumor agents to express strong potency are rather strict and unpredictable. For example, 3xe2x80x2-dephenyl-3xe2x80x2-(2-phenylethenyl)docetaxel, bearing 2-phenylethenyl group instead of the isobutenyl group of RAH-1, has dramatically decreased cytotoxicity ( greater than 20 times) and 3xe2x80x2-dephenyl-3xe2x80x2-neopentyldocetaxel, bearing neopentyl group which has just one more methyl than isobutenyl group, is virtually not cytotoxic against A121 human ovarian, A549 human non-small cell lung, HT-29 human colon and MCF7 human breast cancer cells. While looking at the structure-activity relationships (SAR) of new taxoids that have different substituents at the C-3xe2x80x2 and C-10, we discovered that there are optimum combinations of these two substituents which achieve extraordinarily high activity against drug-resistant cancer cells.
After searching for the best substituent for the C-3xe2x80x2 and the C-10 positions by employing many alkyl groups and alkenyl groups by trial and error, we have identified 1-propenyl, 2-methyl-1-propenyl, 2-methylpropyl, and trifluoromethyl groups to be the optimum substituents for the C-3xe2x80x2 position, and acyl groups, alkoxycarbonyl groups, and N,N-dialkylcarbamoyl groups to be the optimum substituents for the C-10 position.
For example, 3xe2x80x2-dephenyl-3xe2x80x2-(1-propenyl)-10-acetyldocetaxel (Taxoid Ia) showed a substantially better activity spectrum than that of paclitaxel and docetaxel against human ovaian, human non-small cell lung, human colon, and human breast cancer cells mentioned above (see TABLE 1 in EXAMPLE 32). Moreover, this agent possesses 21 times better activity than paclitaxel and 17 times better activity than docetaxel against the drug-resistant human breast cells MCF7-R, which are mammary carcinoma cells 180 fold resistant to a widely used anticancer drug, adriamycin. In the same assay, Holton""s compound RAH-1 showed only marginal activity that was one order of magnitude weaker than that of Taxoid Ia (see TABLE 1 in EXAMPLE 32).
3xe2x80x2-Dephenyl-3xe2x80x2-(2-methyl-1-propenyl)-10-cyclopropanecarbonyldocetaxel (Taxoid IX) showed one order of magnitude better activity than that of paclitaxel and docetaxel against human human breast cancer cells mentioned above (see TABLE 2 in Example 32), and possesses two order of magnitude (142 times) better activity against the drug-resistant human breast cells mentioned above. These extraordinarily high activities are totally unpredictable from the existing SAR studies of paclitaxel and docetaxel, and thus demonstrate the exceptional importance of our discovery.
The taxoids of the formula (I) of this invention are useful for inhibiting tumor growth or regression of tumors in animals including humans and are preferably administered in the form of a pharmaceutical composition including effective amounts of the antitumor agent of this invention in combination with a pharmaceutically acceptable vehicle or diluent.
The pharmaceutical compositions of the antitumor agents of the present invention may be made in any form suitable for desired use, e.g., oral, parenteral or topical administration. Examples of parenteral administration are intramuscular, intravenous, intraperitoneal, rectal, and subcutaneous administration. The vehicle or diluent ingredients should not reduce the therapeutic effects of the antitumor agents of this invention.
Suitable dosage forms for oral use include tablets, dispersible powders, granules, capsules, suspension, syrups, and elixirs. Examples of inert diluents and vehicles for tablets include calcium carbonate, sodium carbonate, lactose and talc. Examples of inert diluents and vehicles for capsules include calcium carbonate, calcium phosphate, and kaolin. Dosage forms appropriate for parenteral administration include solutions, suspensions, dispersions, and emulsions.
The water solubility of the antitumor agents of the formula (I) may be improved by modifying the C-2xe2x80x2 and /or C-7 substituents to incorporate suitable functional groups, such as R3 and R4. In order to increase the water solubility, R3 and R4 can be independently selected from hydrogen and xe2x80x94COxe2x80x94Xxe2x80x94Y, wherein X is selected from xe2x80x94(CH2)nxe2x80x94 (n=1-3), xe2x80x94CHxe2x95x90CHxe2x80x94, cyclohexanediyl, and benzenediyl and Y is selected from xe2x80x94COOH and its pharmaceutically acceptable salts, xe2x80x94SO3H and its pharmaceutically acceptable salts, xe2x80x94NR7R8 and its pharmaceutically acceptable salts, the pharmaceutically acceptable ammonium salt xe2x80x94NR7R8R9, xe2x80x94CONR8R9, or xe2x80x94COOR9, in which
xe2x80x94NR7R8 includes cyclic amine radicals selected from pyrrolidinyl, piperidinyl, morphorino, piperazinyl, and N-methylpiperazinyl;
R7 and R8 are independently selected from hydrogen, allyl, C1-C6 alkyl, and xe2x80x94(CH2)nxe2x80x94Z (n=1-3);
R9 is selected from C1-C6 alkyl, allyl, and xe2x80x94(CH2)nxe2x80x94Z (n=1-3), and
Z is selected from xe2x80x94COOH and its pharmaceutically acceptable salts, xe2x80x94SO3H and its pharmaceutically acceptable salts, xe2x80x94NR7R8 and its pharmaceutically acceptable salts, and pharmaceutically acceptable ammonium salt xe2x80x94NR7R8R10, in which R10 is selected from hydrogen, allyl, and C1-C6 alkyl.
The preparation of the water-soluble analogs of paclitaxel bearing the functionalized acyl groups described above has been disclosed in Kingston et al., U.S. Pat. No. 5,059,699 (1991); Stella et al., U.S. Pat. No. 4,960,790 (1990), the disclosures of which are incorporated herein by reference, and thus it is not difficult for the people in the art to carry out such modifications.